These studies will investigate the importance of the regulation of protein phosphatases to the control of cellular function. Functional interactions between cellular protein phosphatases and their regulators which determine the phosphorylation state and the biological activity of physiologically important proteins will be examined. Experiments designed to elucidate the mode of action of protein phosphatase inhibitor-2 to modulate the activity of protein phosphatase-1 will be undertaken. Studies will focus on the significance of the specific protein structure and the role of phosphorylation of inhibitor-2 in the regulation of protein phosphatase-1. Highly specific antibodies to selected primary sequences will be used to establish the structural determinants essential for inhibitor-2 function. Polyclonal and monoclonal antibodies will be used to study the significance of inhibitor-2 phosphorylation in vitro with purified protein kinases, and in vivo in intact cells in response to physiological stimuli. Protein- chemical and immunological analyses will be undertaken to examine the structural and functional relationship of inhibitor-2 to other phosphatase-1 inhibitors. Antibodies will also be used to examine the molecular and functional properties of inhibitor-2 in other tissues. Synthetic oligonucleotides corresponding to predetermined peptide sequences of inhibitor-2 will be used to clone the cDNA for inhibitor-2. The cDNAs will be used to: (1) generate chimeric gene products for the production of site-specific antisera; (2) produce fragments of inhibitor-2 as either fused or unfused proteins for structure-function studies; (3) identify inhibitor-2 mRNA in tissues, and quantitate the mRNA levels under various metabolic conditions; (4) determine the number of genes coding for inhibitor-2. The ultimate goals of these studies is to provide a clearer understanding of (a) the molecular mechanisms involved in the control of protein phosphatase-1 by its protein modulators, (b) the role of inhibitor-2 in the control of cellular dephosphorylation reactions, and (c) the physiological importance of such regulation to the processes which govern cellular function in mammalian tissues.